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View additional product information for Exosome-Streptavidin Isolation/Detection Reagent - FAQs (10608D)
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我们不推荐这样做，否则链霉亲和素变性时会变为疏水性并发生聚集。
请查看以下可能原因：
•溶液太粘稠。
•蛋白质间相互作用导致磁珠聚集。
尝试以下建议：
•延长分离时间（将管子留在磁力架上2-5分钟）。
•向裂解液中加入DNase I（约0.01 mg/mL）。
•将结合和/或清洗缓冲液中的Tween20浓度增加至约0.05%。
•向结合和/或清洗缓冲液中加入最多至20 mM 的β-巯基乙醇。
选择哪种产品取决于样本的性质、使用的缓冲液和溶液以及下游应用。通常，所有Dynabeads链霉亲和素磁珠都可用于生物素化配体相关的应用；然而，在特殊应用中，有些磁珠可能因其特性而比其他磁珠的效果更好。Dynabeads M-280链霉亲和素磁珠和Dynabeads MyOne链霉亲和素T1磁珠常用于蛋白质和核酸相关应用。Dynabeads M-270链霉亲和素磁珠和MyOne链霉亲和素C1磁珠更适用于核酸诊断，特别是用于含有高浓度高离液盐的样本、涉及小的生物素化抗原的免疫分析以及与BSA不相容的应用，因为这些磁珠没有包被BSA。Dynabeads MyOne链霉亲和素磁珠具有更强的结合能力和更慢的沉降速度，使其适用于自动化应用以及需要分离大量生物素化化合物或其特异性靶标的情况。请点击这里(https://www.thermofisher.com/us/en/home/brands/product-brand/dynal/streptavidin-coupled-dynabeads.html?icid=fr-strep-1)查看选择指南。
您可通过检测上清液中未结合的核酸量，从而得到与Dynabeads链霉亲和素磁珠结合的核酸量。通过OD检测或凝胶电泳可获知核酸浓度。也可以对核酸进行放射性标记以直接在磁珠上检测核酸浓度，或通过荧光标记核酸而在上清液中检测浓度。
我们的Dynabeads 链霉亲和素磁珠可直接用于PCR或real-time PCR。然而，你必须根据经验来优化每一反应体积中所用的磁珠的数量。
尚未检测过每个磁珠结合的链霉亲和素分子精确数量，但每毫克Dynabeads M-280链霉亲和素磁珠大约结合14-16 µg链霉亲和素。
所有生物素试剂都应包含一个间隔臂，至少为一个6-碳接头，以减少空间位阻。这是因为生物素的双环能够进入到链霉亲和素的生物素结合槽深处。一个6碳臂是在生物素和序列第一个碱基之间能够减少空间位阻所需的最短长度。该距离越长，空间位阻越小。6-碳接头是大多数公司采用的标准接头尺寸，应该能够满足大部分应用需求。我们推荐在探针的5’端进行特定的生物素化。
在直接捕获中，生物素化探针/配体先与Dynabeads磁珠偶联，随后再加入样品。在间接捕获中，先将生物素化探针/配体加入到样品中，随后再加入Dynabeads磁珠。直接捕获中预先偶联的配体使得Dynabeads磁珠能够重复使用，而间接捕获在目标分子浓度较低、特异性亲和力较弱或结合动力学速度慢的情况下更有益。请点击以下链接(https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/nucleic-acid-capture-assays.html)，查看捕获图解。
1 mg Dynabeads M-280链霉亲和素磁珠通常可结合650–900 pmol游离生物素、200 pmol生物素化多肽、最多10 μg生物素化抗体、10 μg生物素化双链DNA或200 pmol生物素化单链寡核苷酸。 1 mg Dynabeads M-270链霉亲和素磁珠通常可结合950 pmol以上游离生物素、200 pmol生物素化多肽、最多10 μg生物素化抗体、10 μg生物素化双链DNA或200 pmol生物素化单链寡核苷酸。 1 mg Dynabeads MyOne链霉亲和素C1磁珠通常可结合2,500 pmol以上游离生物素、400 pmol生物素化多肽、最多20 μg生物素化抗体、20 μg生物素化双链DNA或500 pmol生物素化单链寡核苷酸。 1 mg Dynabeads MyOne链霉亲和素T1磁珠通常可结合1,100–1,700 pmol游离生物素、400 pmol生物素化多肽、最多20 μg生物素化抗体、20 μg生物素化双链DNA或400 pmol生物素化单链寡核苷酸。
对于小于1 kb的生物素标记DNA，我们推荐使用Dynabeads M270链霉亲和素磁珠和MyOne C1磁珠。对于大于1kb的双链DNA分子，我们推荐Dynabeads KilobaseBINDER试剂盒。KilobaseBINDER试剂包括M-280链霉亲和素偶联的Dynabeads磁珠和一种含有专利的固定活化剂的结合液，可结合较长的生物素化DNA分子以进行分离。请点击以下链接(https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/immobilisation-of-long-biotinylated-dna-fragments.html)，查看关于长的生物素化DNA片段分离的更多信息。
可以，Dynabeads磁珠可用于分离单链DNA。链霉亲和素Dynabeads磁珠能够以生物素化的DNA片段为靶标，通过使双链DNA变性，从而去除非生物素化链。链霉亲和素偶联的Dynabeads磁珠不会抑制任何酶活性。因此，可以在固相上直接对磁珠结合的DNA进行下一步处理。请点击以下链接(https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/preparing-single-stranded-dna-templates.html)，查看关于单链DNA捕获的更多信息。
磁化率能够衡量磁珠向磁力架迁移的速度，其大小取决于铁含量和氧化铁的特性。Dynabeads磁珠的磁化率是指质量磁化率，单位可以是cgs单位/g或m^3/kg（国际单位制）。对于亚铁磁性和铁磁性物质，质量磁化率取决于磁场强度（H），这些物质的磁化强度与H不是线性关系，而是随着场强增加而趋于饱和。因此， Dynabeads磁珠的质量磁化率是在固定条件下由标准操作程序而测定的。我们产品目录中给出的质量磁化率是国际单位制。磁化率由从高斯（cgs、emu）单位向国际单位的转换，是通过“高斯系数（emu/g或cgs/g）x 4π x 10^-3”而实现的。所得单位也被称为合理化质量磁化率，与（国际单位制）无量纲磁化率单位有所区别。通常，质量磁化率可用来衡量在非均匀磁场中影响物体的力（Fz）。测定Dynabeads磁珠的质量磁化率时，首先对样本称重，然后将样本放置于已知强度的磁场中。随后，再次称重得到样本重量（F1），并与关闭磁场时样本的重量（F0）进行对比。使用下述公式计算磁化率：K x 10^–3 = [(F1-F0) x m x 0.335 x 10^6]，K表示质量为m的样本的质量磁化率。最后，将磁化率转换为国际单位制。
有多种不同的方法可以检测配体与磁珠结合，包括光密度（OD）检测、荧光标记和放射性标记。
对于OD检测，应在配体固定到磁珠上之前检测配体的OD值，并将其与包被后上清液中剩余的配体浓度进行比较。这样可以粗略检测有多少蛋白与磁珠结合。 实验方案： 1.将分光光度计设置到正确的波长。使用偶联缓冲液作为空白组。 2.检测偶联前溶液的吸光值。根据配体的加入量，可能需要进一步稀释以读取吸光值。 3.检测偶联后溶液的吸光值。也可能需要进一步稀释以读取吸光值。 4.计算偶联效率，以“蛋白质摄取量%”表示，如下所示：[(偶联前溶液的吸光值x D) – (偶联后溶液的吸光值x D)] x 100/(偶联前溶液的吸光值 x D)，D = 稀释倍数。 对于荧光标记，我们建议对配体结合量进行反向定量，即检测偶联上清液中剩余的配体量（与原始样本对比），而不是直接检测磁珠上的配体量。将标记的配体加入到磁珠中，并检测上清液中剩余多少配体（而不是结合到磁珠上的配体）。通过与开始时加入的总配体量相比，可以计算出结合到磁珠上的配体量。由于Dynabeads磁珠具有自发荧光，因此，我们不推荐直接检测与磁珠结合的配体的荧光，而是推荐这种间接方法。标记物可以是FITC/PE等。有些研究人员也成功使用了直接检测方法（采用流式细胞仪）。 在3种方法中，放射标记的灵敏度最高，但难度最大。该方法涉及到对配体的一部分进行放射性标记。在偶联前，使用示踪剂量的放射性标记的I-125，将其以一定比例与“冷”配体混合。使用闪烁（γ）计数器对磁珠进行检测，并将磁珠的cpm值与标准品对比，得到磁珠上配体的绝对量。 实验方案： 1.取出适量磁珠，并使用1 mL结合缓冲液清洗。 2.吸取适量人IgG，置于一个单独的管子中。 3.将人IgG与I-125标记的人IgG（30,000–100,000 cpm）混合。 4.使用结合缓冲液将人IgG与I-125标记的人IgG混合物稀释至100mL。 5.室温下孵育30分钟，使用闪烁计数器检测cpm值。 6.清洗磁珠（和包被层）4次，再次检测cpm值。 使用下述方程计算结合率%：（清洗后cpm值/清洗前cpm值）x100%。Dynabeads磁珠有3种尺寸：4.5 µm （M-450）、2.8 µm （M-270/M-280）和1 µm （MyOne beads）。其中最大尺寸的磁珠非常适合细胞等较大的目标，2.8 µm磁珠推荐用于蛋白质组学和分子研究，而最小的1 µm磁珠则适用于自动化处理。
一般来说，在加入配体包被磁珠时，短时间超声是减少磁珠聚集、确保磁珠获得最佳均一性的好方法。一旦目标分子结合到磁珠，就要加倍小心了，以防结合被破坏。链霉亲和素磁珠本身能够承受超声。超声5分钟是可以的，更长时间超声的影响还未被测试。关于链霉亲和素-生物素的相互作用可否被超声破坏目前也尚无相关信息。
只有未包被的环氧基或甲苯磺酰基活化的磁珠可根据需要使用70%乙醇进行清洗除菌。包被的磁珠不可灭菌。
Dynabeads磁珠是一种大小均一、无孔、超顺磁的、单分散的、高度交联的聚苯乙烯微球，整个磁珠由均匀分散的磁性材料构成。该磁性材料由磁赤铁矿(γ-Fe2O3)和磁铁矿(Fe3O4)的混合物组成。在Dynabeads磁珠M-280和M-450中，铁(Fe)分别占磁珠重量的12%和20%。Dynabeads磁珠表面覆盖有一层薄的聚苯乙烯外壳，将磁性材料包裹在内，可防止磁珠泄漏或在内部捕获配体。此外，该外壳也可避免目标分子直接接触铁，同时为每次实验提供特定的表面来吸附或偶联各种分子。
磁珠尺寸和形状均一，确保物理和化学性质稳定一致，进而提高实验结果的质量和可重复性。
Dynabeads磁珠分为3种不同尺寸：4.5 μm （M-450磁珠），2.8 μm （M-270/M-280磁珠）和1 μm （MyOne磁珠）。
可以。请参见此海报（https://tools.thermofisher.com/content/sfs/posters/Exosome-poster-ISEV-2013-Boston.pdf）。
此外，这里还有一些相关引用：
•Blood 91:2573 (1998)
•Science 289:444 (2000)
•J Physiol 537:537 (2001)
•Mol Cell Proteomics 12:587 (2013)
•Biol Reprod 81:717 (2009)
是的，基于不同的膜表面标志物所分离外泌小体中的蛋白表达谱之间会有明显差异。这一结论由Tauro等（http://www.ncbi.nlm.nih.gov/pubmed/23230278）的研究证实，该研究团队基于EpCAM或A33这两种膜表面标志物，从人癌细胞系的条件培养基中分离出两群明显不同的外泌小体。蛋白组学研究结果显示这两群外泌小体是独特的。
我们拥有多款外泌小体分离试剂盒，包括外泌小体—人源CD63（货号10606D），外泌小体—人源CD9（货号10614D），外泌小体—人源CD81（货号10616D）和外泌小体—人源EpCAM，适用于凭借这些常用的外泌小体膜表面抗原来实现外泌小体分离操作。如果您希望使用您的自备抗体通过其他特异性膜表面标志物来分离外泌小体，您也可使用我们的Dynabeads外泌小体免疫沉淀（ProteinA，货号10610D），Dynabeads外泌小体免疫沉淀（ProteinG，货号10612D）或用于分离/检测的外泌小体—链霉亲和素产品（货号10608D）。此外，用户也可选用抗小鼠IgG的Dynabeads磁珠（货号11031或11033）和识别特定膜表面标志物的小鼠单抗来实现外泌小体分离操作。
一般通过流式细胞仪（使用CD9、CD63、TSG101和Alix等膜表面标志物）来鉴定外泌小体，通过EM来研究其形态和尺寸，或凭借LC-MS/MS来实现更为详细的蛋白分析。
这些标志物要基于外泌小体的细胞来源进行选择。最常用于外泌小体分离和鉴定的膜表面标志物为CD9、CD63、CD81或TSG101。下表列举了一些最近用于鉴定或分离外泌小体的参考文献和膜表面标志物：
膜表面标志物
参考文献
Alix, CD63, EpiCam, HSP70, TSG101
Mol Cell Proteomics 12:587 (2013)
CD9, CD63
Hum Mol Genet 21:R125 (2012)
CD63, MHC II
J Biol Chem 278:52347 (2003)
CD9, CD81, Lamp1, TSG101
Cancer Res 67:7458 (2007)
CD63
Nature Cell Biol 9:654 (2007)
Alix, CD37, CD53, CD63, CD81, CD82, TSG101
J Cell Biol 200:373 (2013)
CD59, CD63, CD133, TSG101
FASEB J 23:1858 (2009)
除沉淀法之外，外泌小体还可通过超速离心或密度梯度分离法实现分离。用户也可使用靶向外泌小体标志物（例如人源的CD9、CD63、CD81、EpCAM）的Dynabeads磁珠或二抗包被的Dynabeads磁珠（使用靶向其他外泌小体膜表面标志物的不同抗体），凭借磁性方法来分离外泌小体。
外泌小体被报道具有多种不同的功能，如抗原呈递、凋亡、血管发生、炎症和凝血作用，这些作用是通过蛋白/脂类交换或信号途径的激活来实现的。外泌小体提供了一种胞间遗传物质交换的全新机制，并能够介导细胞间的相互通讯。外泌小体也能够转运和传播传染性物质，如朊蛋白和逆转录病毒。
外泌小体是微小的卵形或杯形膜结构，大小在30-150 nm，其中包含有mRNA，microRNA，蛋白和脂类。外泌小体可由正常，异常或肿瘤细胞释放进入血、尿、唾液和乳汁等体液中。外泌小体源于内吞型细胞器，并作为与质膜融合的多泡体（MVB）而从细胞释放出来(J Cell Biol 200:373 (2013)).
We do not recommend this as streptavidin becomes hydrophobic and aggregates during denaturation.
Please review the following possibilities for why your Dynabeads magnetic beads are not pelleting:
- The solution is too viscous.
- The beads have formed aggregates because of protein-protein interaction.
Try these suggestions:
- Increase separation time (leave tub on magnet for 2-5 minutes)
- Add DNase I to the lysate (~0.01 mg/mL)
- Increase the Tween 20 concentration to ~0.05% of the binding and/or washing buffer.
- Add up to 20 mM beta-merecaptoethanol to the binding and/or wash buffers.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Which product to choose depends on the properties of your sample, the buffers and solutions applied, as well as the downstream application. In general, all Dynabeads Streptavidin beads can be used in applications involving biotinylated ligands; however, some beads may perform better than others in particular applications due to their characteristics. Dynabeads M-280 Streptavidin beads and Dynabeads MyOne Streptavidin T1 beads are commonly used for protein and nucleic acid applications. Dynabeads M-270 Streptavidin beads and MyOne Streptavidin C1 beads are preferred for nucleic acid diagnostics, specifically with samples that have a high concentration of chaotropic salts, immunoassays involving small biotinylated antigens, and in applications that are not compatible with BSA, as these beads are not blocked with BSA. Dynabeads MyOne Streptavidin beads offer increased binding capacity and slower sedimentation rate, making them ideal for automated applications and when larger amounts of a biotinylated compound or its specific target need to be isolated. Please see the selection guide here ( https://www.thermofisher.com/us/en/home/brands/product-brand/dynal/streptavidin-coupled-dynabeads.html?icid=fr-strep-1).
You can assay the supernatant for unbound nucleic acid to determine the amount of nucleic acid bound to the Dynabeads Streptavidin beads. The concentration of nucleic acids can be checked by measuring the OD or by running them on a gel. Alternatively, the nucleic acids can be labeled radioactively and the concentration measured directly on the beads, or fluorescently and measured in the supernatant.
Our Dynabeads Streptavidin magnetic beads can be used directly in PCR or real-time PCR. However, you would have to empirically optimize the amount of beads to be used per volume of reaction.
The exact number of streptavidin molecules bound per bead is not measured, but is approximately 14-16 µg streptavidin per milligram Dynabeads M-280 Streptavidin magnetic beads.
All biotin reagents should contain a spacer arm, at least a 6-carbon linker, to reduce steric hindrance. This is because the bicyclic ring of biotin goes deep into the biotin binding cleft in streptavidin. A 6-carbon arm is the minimum length between biotin and the first base of sequence that is required to reduce the steric hindrance effect. The longer this distance is, the less the steric hindrance. A 6-carbon linker is standard linker size from most companies and should be enough for most applications. We recommend specific biotinylation at the 5'-end of the probe.
In direct capture, the biotinylated probe/ligand is first coupled to the Dynabeads magnetic beads followed by addition of your sample. In indirect capture, the biotinylated probe/ligand is first added to the sample followed by addition of your Dynabeads magnetic beads. Precoupled ligand for direct capture allows you to reuse the Dynabeads magnetic beads, while an indirect approach can be beneficial when the concentration of your target is low, specific affinity is weak, or the binding kinetics is slow. Please see the following link (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/nucleic-acid-capture-assays.html) for a diagram of the capture.
- One milligram of Dynabeads M-280 Streptavidin magnetic beads typically binds 650-900 pmol of free biotin, 200 pmol of biotinylated peptide, up to 10 µg of biotinylated antibody, 10 µg of biotinylated double-stranded DNA, or 200 pmol of biotinylated single-stranded oligonucleotides.
- One milligram of Dynabeads M-270 Streptavidin magnetic beads typically binds more than 950 pmol of free biotin, 200 pmol of biotinylated peptide, up to 10 µg of biotinylated antibody, 10 µg of biotinylated double- stranded DNA, or 200 pmol of biotinylated single-stranded oligonucleotides.
- One milligram of Dynabeads MyOne Streptavidin C1 magnetic beads typically binds more than 2,500 pmol free biotin, 400 pmol of biotinylated peptides, up to 20 µg of biotinylated antibody, 20 µg of biotinylated double-stranded DNA, or 500 pmol of biotinylated single-stranded oligonucleotides.
-One milligram of Dynabeads MyOne Streptavidin T1 magnetic beads typically binds 1,100-1,700 pmol free biotin, 400 pmol of biotinylated peptides, up to 20 µg of biotinylated antibody, 20 µg of biotinylated double- stranded DNA, or 400 pmol of biotinylated single-stranded oligonucleotides.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
For biotin-labeled DNA that is less than 1 kb, we recommend you use Dynabeads M270 Streptavidin (Cat. No. 65305) and MyOne C1 magnetic beads (Cat. No. 65001). We recommend our Dynabeads KilobaseBINDER Kit (Cat. No. 60101), which is designed to immobilize long (>1 kb) double-stranded DNA molecules. The KilobaseBINDER reagent consists of M-280 Streptavidin-coupled Dynabeads magnetic beads along with a patented immobilization activator in the binding solution to bind to long, biotinylated DNA molecules for isolation. Please see the following link (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/immobilisation-of-long-biotinylated-dna-fragments.html) for more information in regards to long biotinylated DNA fragment isolation.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Yes, Dynabeads magnetic beads can be used to isolate single-stranded DNA. Streptavidin Dynabeads magnetic beads can be used to target biotinylated DNA fragments, followed by denaturation of the double-stranded DNA and removal of the non-biotinylated strand. The streptavidin-coupled Dynabeads magnetic beads will not inhibit any enzymatic activity. This enables further handling and manipulation of the bead-bound DNA directly on the solid phase. Please see the following link (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/preparing-single-stranded-dna-templates.html) for more information in regards to single-stranded DNA capture.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Magnetic susceptibility is a measure of how quickly the beads will migrate to the magnet. This will depend on the iron content and the character of the iron oxide. The magnetic susceptibility given for the Dynabeads magnetic beads is the mass susceptibility, given either as cgs units/g or m^3/kg (the latter being an SI unit). For ferri- and ferromagnetic substances, the magnetic mass susceptibility is dependent upon the magnetic field strength (H), as the magnetization of such substances is not a linear function of H but approaches a saturation value with increasing field. For that reason, the magnetic mass susceptibility of the Dynabeads magnetic beads is determined by a standardized procedure under fixed conditions. The magnetic mass susceptibility given in our catalog is thus the SI unit. Conversion from Gaussian (cgs, emu) units into SI units for magnetic mass susceptibility is achieved by multiplying the Gaussian factor (emu/g or cgs/g) by 4 pi x 10^-3. The resulting unit is also called the rationalized magnetic mass susceptibility, which should be distinguished from the (SI) dimensionless magnetic susceptibility unit. In general, magnetic mass susceptibility is a measure of the force (Fz) influencing an object positioned in a nonhomogenous magnetic field. The magnetic mass susceptibility of the Dynabeads magnetic beads is measured by weighing a sample, and then subjecting the sample to a magnetic field of known strength. The weight (F1) is then measured, and compared to the weight of the sample when the magnetic field is turned off (F0). The susceptibility is then calculated as K x 10^-3 = [(F1-F0) x m x 0.335 x 10^6], where K is the mass susceptibility of the sample of mass m. The susceptibility is then converted to SI units.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
There are different methods to check binding of ligands to the beads, including optical density (OD) measurement, fluorescent labeling, and radioactive labeling.
For OD measurement, you would measure the OD of the ligand before immobilization to the beads and compare it with the ligand concentration that is left in the supernatant after coating. This gives a crude measurement of how much protein has bound to the beads.
Protocol:
1.Set spectrophotometer to the right wavelength. As a blank, use the Coupling Buffer.
2.Measure the absorbance of the Pre-Coupling Solution. A further dilution may be necessary to read the absorbance, depending upon the amount of ligand added.
3.Measure the absorbance of the Post-Coupling Solution. A dilution may be necessary to read the absorbance.
4.Calculate the coupling efficiency, expressed as the % protein uptake, as follows. [(Pre-Coupling Solution x D) - (Post-Coupling Solution x D)] x 100/(Pre-Coupling Solution x D) where D = dilution factor.
For fluorescent labeling, we suggest negatively quantifying the amount of ligand bound by measuring ligand remaining in the coupling supernatant (compared to the original sample), rather than directly measuring the ligands on the beads. Add labeled ligand to the beads, and measure how much ligand is left in the supernatant (not bound to the beads). By comparing this with the total amount added in the first place, you can then calculate how much of the ligand that has been bound to the beads. Keep in mind that the Dynabeads magnetic beads are also autofluorescent, which is why direct measuring of fluorescence of the bead-bound ligands is not recommended, but rather this indirect approach. The label could be, for example, FITC/PE. Some researchers perform a direct approach with success (using a flow cytometer).
Radioactive labeling is the most sensitive method of the three, but it is also the most difficult one. It involves radioactively labeling a portion of the ligand. We use radiolabeled I-125 in tracer amounts and mix it with "cold" ligands in a known ratio before coupling. The absolute quantities for the ligand on the beads should be obtained by measuring the beads in a scintillation (gamma) counter and comparing the cpm with a standard.
Protocol:
1.Take out an appropriate amount of beads and wash the beads in 1 mL of binding buffer.
2.Pipette out desired amount of human IgG in a separate tube.
3.Mix the human IgG with I-125-labeled human IgG (30,000 - 100,000 cpm).
4.Dilute the mixture of human IgG and I-125-labeled human IgG to 100 mL in binding buffer.
5.Incubate for 30 minutes at room temperature and measure the cpm in a scintillation counter.
6.Wash the beads (with coating) four times, and measure cpm again.
The % binding is calculated by using the equation : (cpm after washing/cpm before washing)x100%.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Dynabeads magnetic beads come in three sizes: 4.5 µm (M-450), 2.8 µm (M-270/M-280), and 1 µm (MyOne beads). The largest of the Dynabeads magnetic beads is ideal for big targets like cells. The 2.8 µm beads are recommended for proteomics and molecular applications. The smallest of the beads, 1 µm, are ideal for automated handling.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
In general, short sonication is a good way to reduce aggregation of the beads and ensure optimal homogenous conditions at the time of ligand addition when coating the beads. When target is bound to the beads, more care is needed, as the binding might break. The streptavidin beads themselves should tolerate sonication. We have not tested sonication for long periods, but 5 minutes is fine. We do not have information about the streptavidin-biotin interaction being broken by such treatment.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
If desired, the uncoated epoxy or tosylactivated beads can be sterilized by washing with 70% ethanol. Coated beads cannot be sterilized.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Dynabeads magnetic beads are uniform, non-porous, superparamagnetic, monodispersed and highly cross-linked polystyrene microspheres consisting of an even dispersion of magnetic material throughout the bead. The magnetic material within the Dynabeads magnetic beads consists of a mixture of maghemite (gamma-Fe2O3) and magnetite (Fe3O4). The iron content (Fe) of the beads is 12% by weight in Dynabeads magnetic beads M-280 and 20% by weight in Dynabeads magnetic beads M-450. The Dynabeads magnetic beads are coated with a thin polystyrene shell which encases the magnetic material, and prevents any leakage from the beads or trapping of ligands in the bead interior. The shell also protects the target from exposure to iron while providing a defined surface area for the adsorption or coupling of various molecules.
Uniformity of bead size and shape provides consistent physical and chemical properties. These uniform physical characteristics lead to high-quality, reproducible results.
The Dynabeads magnetic beads are available in three different sizes: 4.5 µm (M-450 beads), 2.8 µm (M-270/M-280 beads) and 1 µm (MyOne beads).
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
For the short-term, exosomes can be stored at 4 degrees C for up to 1 week. For the long-term, exosomes can be stored at -20 degrees C or -80 degrees C. When storing exosomes for the long term, it is important to consider whether they will need to be thawed more than once for the target application. If multiple applications (and thus multiple thaws) will be used for analysis, then we recommend aliquoting the exosome resuspensions into multiple tubes so that each tube will only undergo one freeze/thaw cycle. We have found that multiple freeze thaw cycles can cause damage to the exosomes and reduce their numbers.
Unlike serum, plasma contains numerous clotting factors and some additional proteins that can make it difficult to work with. We‘ve provided two protocol options, one with proteinase K (PK) and one without, in order to ease this difficulty. The protocol using PK is most useful when the end goal is analysis of the RNA or protein cargo contained inside the exosomes. It can also be used to isolate exosomes for use in other downstream applications, but it is most useful for RNA and protein analysis. The protocol without PK also isolates good quality exosomes, just not quite as pure as the PK protocol. The “no PK” protocol is more useful for isolating exosomes that will be used for surface protein analysis or electron microscopy identification.
There are several possible reasons why Western blotting analysis is challenging:
1. Not enough sample volume added. Exosomes can contain a fairly low amount of protein cargo, so for an initial experiment we recommend adding as much of the sample as possible.
2. Antibody concentration should be titrated. Also, they should ideally be used fresh and need to be stored properly.
3. Depending on the exosomal surface marker, certain gel conditions might be more optimal for the target antibody (e.g., reducing/nonreducing and denaturing/nondenaturing). We suggest checking with the manufacturer and exosome community about which Western blotting conditions are recommended for the specific marker you are targeting and the specific antibody you are using.
4. General Western techniques. Westerns can be tricky so we recommend the use of a positive control for initial testing to make sure the entire workflow is functioning as it should. Any protein or antibody can be used as long as they meet the conditions you need (e.g., denaturing vs. non-denaturing). In addition, when picking the protein, try to steer clear of those that are present at very high or very low concentrations in your sample to prevent overloading the blot or total absence of signal.
This can vary depending on the sample type, volume of sample, isolation method, and exosome content/concentration. Listed below are some examples:
1) When exosomes are isolated from 30 mL of HeLa cell culture medium using the Total Exosome Isolation Reagent, it is possible to recover approximately 8 ng exosomal RNA.
2) For exosomes recovered from 4 mL serum, approximately 2 ng exosomal RNA can be obtained.
In both cases, these amounts of RNA are sufficient for RNA library prep for Ion PGM or Ion Proton sequencing. For real-time PCR analysis, substantially smaller amounts of RNA are needed and much lower sample volumes can be used. For example, RNA recovered from 3 µL serum or 30 µL medium is enough for one qRT-PCR reaction.
No, the described effect does not have a negative impact on the RNA recovery.
The Total Exosome RNA & Protein Isolation Kit (Cat. No. 4478545), developed specifically for exosome samples, uses an initial acid-phenol:chloroform extraction followed by a final purification over a glass-fiber filter column to provide a robust initial RNA purification. After the acid-phenol extraction, ethanol is added to the aqueous phase and then passed through a filter cartridge containing the glass-fiber filter, which immobilizes the RNA. The filter is washed, and the RNA is eluted with a low ionic-strength solution.
The kit recovers all RNA longer than approximately 10 nt through several kb, although the majority of RNA contained in purified exosomes ranges in size from 20-300 nt. The kit also contains an additional protocol for enrichment of short RNA (less than 200 nt), but we recommend the total RNA isolation protocol in order to maximize recovery of all RNA, including vario+us mRNA, rRNA, and ncRNA fragments.
This kit also provides an option to recover protein from the same sample through the use of the Exosome Resuspension Solution.
The kit can be used to isolate RNA and protein from exosomes purified from any sample type, using either the Total Exosome Isolation reagents or any other protocol such as ultracentrifugation.
Yes. We have successfully recovered exosomes from up to 5 mL of CSF using the procedure listed in the Total Exosome Isolation (from other body fluids) manual (Cat. No. 4484453).
Yes, overnight precipitation at 4 degrees C is acceptable for urine, CSF, and amniotic fluid without sacrificing quality or yield.
For larger sample volumes than those recommended in the manuals, a longer centrifugation is recommended to ensure maximum recovery of the exosomes. The exact time will depend on several factors including the rotor, the sedimentation coefficient of the exosomes, size of the tube, sample volume and type, and centrifugation speed, and should be determined empirically. For example, for 5-10 mL sample volumes, 1 hr of centrifugation at 10,000 x g is sufficient to pellet the exosomes.
Here are some examples:
HeLa cells grown to approximately 2 x 10e7 per T175 flask in 30 mL medium with exosome-depleted FBS. From 1 mL of this medium, one can recover approximately 4-8 x 10e9 exosomes using the Total Exosome Isolation Reagent (from cell culture media), as measured with Nanosight LM10 instrument. These numbers will be somewhat different for different cell lines and whether exosome-depleted FBS or no FBS or synthetic medium is used.
From 100 µL serum one can recover approximately 1.5-3 x 10e11 exosomes using Total Exosome Isolation Reagent, as measured with Nanosight LM10 instrument.
Here is a paper put together by our scientists that compares our exosome isolation protocol with standard ultracentrifugation protocols, with respect to RNA profiling: https://www.wjgnet.com/2222-0682/full/v3/i1/11.htm
For each reagent, the minimal volume tested can be found listed in the reagent manual. For most body fluids the minimum volume tested is 100-200 µL, but it is slightly larger for urine (800 µL) and cell culture media (1 mL). Smaller volumes can be used, especially for serum and plasma, but we‘ve found that the minimums listed in the manuals and above provide a usable amount of exosomes for multiple downstream applications.
Serum and plasma contain a very high number of exosomes, thus the pellet is visible even if you isolate exosomes from as little as 100 µL. Other body fluids, such as urine or cell culture media, have significantly lower concentrations of exosomes, and the pellet is often not visible after centrifugation. Since the pellet sticks very tightly to the tube, it is okay to remove the supernatant completely prior to resuspending in PBS. If needed, marking the tube so that you know where the pellet will adhere upon centrifugation. It is absolutely crucial to remove the supernatant completely. If you don‘t, there will be a significant amount of the reagent left, and when you resuspend the exosomes, some of them might still be in the form of aggregates at the bottom of the tube.
The reagent does not bind the exosome surface, and only trace amounts remain in the exosome pellet after isolation, so it should not interfere with downstream biological studies. However, it is important to remove the supernatant completely, prior to resuspending the exosome pellet in PBS or other buffer of choice. In case there are still concerns regarding trace amounts of the reagent being present, they can be removed by dialysis or using Exosome Spin Columns (MW 3000) (Cat. No. 4484449).
We currently have a total of five reagents that allow isolation of exosomes from major body fluids: serum, plasma, urine, “other body fluids” (CSF, saliva, milk, ascitic fluid, amniotic fluid), and cell culture media. All of these reagents share the same core compound, but they and their protocols have been carefully optimized to enable efficient isolation of exosomes from the specific sample type.
Plasma is a more challenging type of sample compared to serum, as it has high levels of clotting factors, which can be more problematic. The current serum reagent will work on plasma, but the preparation will likely contain more contaminating proteins and microvesicles. For plasma, we recommend using the Total Exosome Isolation reagent (from plasma) (Cat. No. 4484450), as the reagents and protocol have been specifically optimized to handle plasma and its different composition.
Yes, the reagents can also be used with mouse samples. Presumably, the reagents can also isolate exosomes from samples of any species, but they have only been tested with human and mouse.
The obtained sample contains all exosomes, with insignificant amounts of some other microvesicles, and large protein molecules/complexes that have been co-precipitated (in the case of serum and some of the other body fluids). This purity level works fine for most applications and is balanced by the method benefits which include a fast and simple workflow, no need for special equipment (such as an ultracentrifuge), complete recovery of exosomes, flexibility to work with small sample volumes (e.g., 100 µL), and the capability to process multiple samples in one experiment.
For ultra-pure exosomes from cell culture media, we have available Dynabeads magentic beads decorated with anti-CD63 antibodies, which allow recovery of a very clean population of exosomes, following initial purification with the Total Exosome Isolation Reagent (from cell media). Workflow time will increase and the final yield of the exosomes will be lower, but for projects that require an ultra-clean population of CD63-positive exosomes from cell culture media this is the best option.
Dynabeads magnetic beads complexed with streptavidin (Cat. No. 10608D) are also available for use with a customer‘s own biotinylated antibodies specific for their exosome sub-population of choice. These products can be used not only for isolation of highly pure exosome sub-populations, but also allow detection of exosomes with flow cytometry, something that has been extremely difficult to achieve due to their small size.
We believe that these products are currently the best options for exosome isolation, but as the definition of exosomes solidifies and the demands of the field of microvesicle research become clearer, we can focus on additional products
Isolation of exosomes is presently a tedious, non-specific, and difficult process. In the course of development of reagents for isolation of exosomes, we evaluated many different technologies, including “gold standard” ultracentrifugation, ultrafiltration; gel-filtration columns, HPLC, and filters. In addition to these simpler methods, we evaluated more advanced approaches including precipitation using various polymers, and bead and column binding using antibodies and various lectins. We also evaluated commercially available products from System Biosciences and other companies. After evaluation, we selected one of the polymers, based on its superior performance, which became the key component of the Total Exosome Isolation reagents (patent application filed). By tying up water molecules, the reagent forces less-soluble components, such as vesicles, out of solution, which allows them to be collected by a short, low-speed centrifugation. The recovered exosomes are then ready for either biological studies or end-point analysis.
Traditional isolation of exosomes from cell culture media and body fluids is a tedious and difficult process with the most widely used approach based on ultracentrifugation in combination with sucrose density gradients or sucrose cushions to float the relatively low-density exosomes away from other vesicles and particles. These protocols can range in time from 8 to 30 hrs and require an ultracentrifuge and extensive training to ensure successful isolation of exosomes. Despite these drawbacks, ultracentrifugation is still the most popular approach for exosome isolation. However, within the last 2 years, several exosome isolation reagents for cell culture media and various body fluids have become commercially available. Thermo Fisher Scientific Total Exosome Isolation reagents allow recovery of exosomes using a very short and reliable protocol which is becoming more and more popular.
In addition to their 30-150 nm size, to be categorized as exosomes, currently, the vesicles should be positive for certain surface protein markers, such as tetraspanins. The most widely accepted marker is CD63, but CD81, CD9 are utilized as well. Western blotting for these targets on the sample of interest is a relatively simple way to confirm the vesicles are exosomes. However, the current definition of exosomes is not set in stone as there is no absolute consensus in the field. It will probably take another several years for the field to agree on the exact specification and nomenclature for all nano- and micro- vesicles including exosomes.
At the moment there are only two straightforward options to determine the concentration of exosomes in a sample: the NanoSight instrument and the Izon instrument. The Nanosight instrument enables counting and sizing of nanoparticles (10-1000 nm size) using light scattering and brownian motion, while the Izon instrument accomplishes the same thing using nanopore analysis.
Exosomes, at 30-150 nm, are too small to be seen using a regular microscope, which is limited to objects that are at least several micrometers in size. The typical methods of analysis for size distribution include the Nanosight instrument and electron microscopy. Although very different in methodology, both technologies allow one to study nanoparticles as small as 10 nanometers in size.
Exosomes are classically described as vesicles originating from the endosomal compartment through fusion of multivesicular bodies with the plasma membrane. They are a part of a larger family of vesicles secreted by cells, including microvesicles, ectosomes, and shed particles, which originate by direct budding from the plasma membrane. It is extremely challenging to separate these entities using currently available techniques and instruments due to overlap in their size, density, and overall similar composition.
Exosomes are tiny vesicles (30-150 nm) containing protein and/or RNA cargo within a lipid bi-layer membrane. Exosomes can differ extensively in both their cargo and surface proteins, and different cell types can secrete different, sometimes multiple, types of exosomes.
The current definition of exosomes is complex as no absolute consensus has been made in the research field. Typically, exosomes are defined as vesicles floating in sucrose solution at a density of approximately 1.13 to 1.19 g/mL during ultracentrifugation-based isolation with an expected size of 30-150 nm based on electron microscopy analysis. Exosomes can also be defined and identified by their surface protein markers, which include: tetraspanins (CD63, CD81, CD9) and others like ALIX. Currently, we don‘t have the appropriate tools or enough knowledge to create a clear and simple definition of exosomes that would differentiate them from other micro/nanovesicles.
Ultracentrifugation typically recovers fewer exosomes compared to the Total Exosome Isolation reagent. Since cell culture medium has very low exosome content, the exosome pellet is invisible in many cases. Assuming that you have not lost the pellet, you can do Nanosight analysis or use some other easy readout to confirm how much you have isolated.
Sometimes exosomes stick together upon isolation with the reagent or by ultracentrifugation. They don‘t fuse, but are just clumped together. You can (1) add buffer such as PBS to the exosome pellet, let the sample sit at RT for several hours, then pipet the sample extensively up and down, or (2) gently vortex the pellet.
We offer the Total Exosome RNA and Protein Purification Kit (Cat. No. 4478545) for isolation of total RNA and protein from exosomes.
We typically count exosomes with the Nanosight LM 10 instrument, rather than measure their weight. To provide an idea about numbers, when HeLa cells were grown to approximately 2 x 10e7 cells per T175 flask in 30 mL cell culture medium in the presence of exosome-depleted FBS, 1 mL of this cell medium yielded approximately 4-8 x 10e9 exosomes isolated with the reagent. The number will be somewhat different depending on the cell line or medium used.
We recommend using the Qubit 2.0 Fluorometer to easily measure the signal associated with exosomes post-labeling.
Please review the following options:
- You can normalize samples using miR-16, miR-24, 18S rRNA, or GAPDH, which are all highly abundant in serum-derived exosomes.
- If you have enough biological replicates and a reliable workflow (sample prep to RT to qPCR) and minimal error bars, you can skip normalization and instead, use the same sample volume input.
We recommend resuspending the exosomes in PBS. Ideally, the isolations should be performed with a lower volume of serum, e.g., 100 µL, so that the exosome pellet is small and easier to resuspend. This sample should contain a substantial amount of RNA and protein for typicaly downstream assays, including Western blotting and Real-Time PCR. If the pellet is large and difficult to resuspend, (e.g., if the isolation was done from greater than 1 mL of serum), let it sit in PBS for 1 hour at room temperature followed by pipetting or gently vortexing.
In our experience, there is no difference between exosome isolation from fresh serum and frozen serum, stored at -20 degrees C or -80 degrees C. However, multiple (greater than 5) freeze/thaw cycles can damage the exosomes.
The Total Exosome Isolation reagent precipitates exosomes by tying up water molecules and forcing them out of solution. Once the reagent is completely removed and PBS is added, exosomes will start going back into solution. Typically, no washing is needed. You can collect the remaining droplets of the reagent by addtiional quick centrifugation, and discard them. To do a wash, we would recommend performing a quick rinse in PBS by spinning for 5 minutes at 14,000 g at 4 degrees C. We do not recommend vortexing of the pellet.
The easiest way to count exosomes is by Nanosight analysis (e.g., Nanosight LM10). With most of these instruments, you can obtain valuable information on their size distribution and count. With electron microscopy, you will be able to see just a few “zoomed in” vesicles and see their size and shape, but you will not be able to quantify exosomes.
There are typically no problems with contamination when using exosomes (recovered with the Total Exosome Isolation reagent) in downstream biological assays.
We would recommend using 20-50 mL of cell culture medium and 4-8 mL of serum or plasma. You can use the Total Exosome Isolation reagent to recover the exosomes. Ultracentrifugation is also an option, but yields will be lower.
Yes, exosomes have a very diverse RNA and protein content. RNA species, in addition to mRNA, include miRNA, rRNA, tRNA, and many non-coding RNAs, some of which do not map to the databases. Most of the exosomal RNA “cargo” is short, 20-200 nt (including mRNA fragments), but some mRNA molecules are full-length, up to several kb long.
No, exosomal RNA has about the same stability as RNA recovered from other sources. One important thing to note: since exosomes contain very low amounts of RNA, the concentration of isolated exosomal RNA in the tube is often is very low, resulting in very poor stability. We recommend taking extra precaution to avoid RNase contamination by using RNase-free water, buffers, tips, tubes, etc., and storing the RNA stock at -20 degrees C or below, in aliquots, at the highest possible concentration.
When you harvest the cell culture medium, it should be spun down to remove cells and debris. You can then keep this “clarified” medium at 4 degrees C for up to one week, or isolate exosomes by precipitation with the Total Exosome Isolation reagent or other techniques.
We recommend using our Total Exosome RNA and Protein Isolation Kit (Cat. No. 4478545), which has reagents enabling splitting of the exosomes and recovery of either their protein cargo or RNA cargo (via column based purification). Overall, exosomes are similar to cells in terms of membrane composition, and a number of commonly used sample prep kits will work fine on exosomes as well.
We recommend using anti-mouse IgG HRP, since it will not recognize the heavy or light chains of the antibodies eluted off the beads.
Exosomes can be vortexed briefly without damaging their membrane, but there are no detailed studies on the effects of prolonged vortexing or sonication on exosome integrity.
We would not recommend multiple (greater than 5) freeze/thaw cycles, as that would damage the exosomes. Ideally, we would recommend freezing the exosomes in single-use aliquots.
Prior to Western blotting, exosomes can be pre-enriched followed by lysis using lysis buffers such as RIPA, NP40, or Triton, together with protease inhibitors. This is followed by a short vortex or sonication in order to have complete lysis. For detection by Western blotting, the pre-enriched exosomes should be lysed in 5X RIPA, for example. We have tried the following: Mix 7.5 µL pre-enriched exosomes + 1.9 µL 5X RIPA w/ protein inhibitors, followed by a short sonication and incubation on ice for 15 mins. Take 9.4 µL lysed exosomes + 9.4 µL 2X SDS (Laemmli) + 1 µL loading buffer and incubate 5 min at 95 degrees C and add all to one well. Given that the pre-enrichement step was successful, this should ensure maximum loading of exosomes on the gel and detection by Western blotting.
If the pre-enriched exosomes have been further isolated using magnetic beads to pull out subpopulations of exosomes, these can also be lysed using 1X RIPA on ice followed by Laemmli and loading buffer in the same way. The beads can either be removed prior to gel loading using a magnet or apply the lysed exosomes and beads on the gel. The beads will be left in the well while the target moves in the gel.
Please see our protocols, including Western blotting:
- Schageman J., Zeringer E., Li M., Barta T., Lea K., Jian Gu, Magdaleno S., Setterquist R., and Vlassov A.V. The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo. BioMed Research International. 2013, 2013:253957. doi: 10.1155/2013/253957.
- Zeringer E., Li M., Barta T., Schageman J., Pedersen K.W., Neurauter A., Magdaleno S., Setterquist R., Vlassov A.V. (2013) Methods for the extraction and RNA profiling of exosomes. World J Methodology. 3, 11-18.
While different cells react differenty under different conditions, in general, if you grow cells in FBS-free media, fewer exosomes will be secreted as cells are starved. Some cells, such as HeLa cells, are more durable under exosome-depleted FBS conditions. We have been able to isolate 4-8 x 10E9 exosomes from 1 mL of this cell culture medium. For more information, please refer to the following reference: Schageman J. et al., BioMed Research International, Volume 2013 (2013), Article ID 253957.
A pure sample of exosomes contains very low amounts of RNA. Therefore, the best way to increase yeild is to scale up the isolation. For cell culture media, you can try starting with 100 mL of sample.
Yes, please check our application note at https://tools.thermofisher.com/content/sfs/brochures/Exosome%20Tracing_App%20Note.pdf that lists protocols employing SYTO RNASelect stain (Cat. No. S32703).
There are several typical reasons why Western blot analysis does not work:
1. Not enough sample volume added. Exosomes can contain a fairly low amount of protein cargo, so for an initial experiment we recommend adding as much of the sample as possible.
2. Antibodies are not optimal. We suggest testing antibodies (e.g., anti-CD63 or other exosomal marker) from 2-3 manufacturers, carefully checking what concentration is recommended. Also, they should ideally be used fresh, and need to be stored properly.
3. Depending on the exosomal surface marker, certain gel conditions might be more optimal for the target antibody (e.g., reducing/nonreducing and denaturing/nondenaturing). We suggest checking with the manufacturer and exosome community about which Western blot conditions are recommended for the specific marker you are targeting and the specific antibody you are using.
4. General western techniques. Westerns can be tricky, so we recommend the use of a positive control for initial testing to make sure the entire workflow is functioning as it should. Any protein or antibody can be used as long as they meet the conditions you need (e.g., denaturing vs. non-denaturing). In addition, when picking the protein, try to steer clear of those that are present at very high or very low concentrations in your sample to prevent overloading the blot or total absence of signal.
You can add the loading buffer directly to the exosome pellet, heat, and load onto your gel for downstream western analysis. There is no need to air dry the pellet. 1 mL cell culture medium per lane is a good starting point, but may need to be optimized based on the protein of interest, antibody used, etc.
When you isolate exosomes from cell culture medium, the pellet is invisible in most cases, unless you are using a large volume (e.g., 30 mL). With serum, plasma, and other body fluids, the pellet is typically visible if you process over 100 µL of sample.
While we have not tested ESC, for those media that we have tested, the number of secreted exosomes and isolation protocols are very similar. When growing cells, use chemically defined media or exosome-depleted FBS. Exosomes can be collected at 24 hours. 1-5 mL of media is a good starting point to isolate exosomes. This should be enough for several real-time PCR or Western blotting experiments. Sequencing and some other downstream analyses may require more starting material, and for this, we would recommend 30 mL cell culture medium to start.
Please refere to the following reference: The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo. Jeoffrey Schageman, Emily Zeringer, Mu Li, Tim Barta, Kristi Lea, Jian Gu, Susan Magdaleno, Robert Setterquist, and Alexander V. Vlassov. Hindawi Publishing Corporation, BioMed Research International. Volume 2013, Article ID 253957, 15.
Overall, we have not encountered issues related to hemolysis. We would recommend using care with body fluid collection and preservation. For recommendations on standardization of sample collection, isolation, and analysis, please refer to the following reference: Witwer et al., Journal of Extracellular Vesicles (2013) 2:20360.
Overall, the exosomes recovered from cell culture media are very clean. Exosomes derived from body fluids sometimes have a few contaminating microvesicles and large protein aggregates. The recovered exosomes can be analyzed by Nanosight (size distribution and count) or electron microscopy. Western analysis could be performed using protein markers, including CD63, CD81, CD9, Alix, or Annexin. RNA cargo can be analyzed by real-time PCR (e.g., of let7).
We routinely use the Total Exosome RNA and Protein Isolation kit (Cat. No. 4478545), but there are a number of other kits that are popular among researchers, including mirVana isolation kits and TRIzol reagent. For analysis, Real-Time PCR with TaqMan miRNA assays would be our recommendation.
Apoptotic bodies are large (greater than 800 nm), so they will mostly be removed from the sample during pre-spin (along with cells and debris). The Total Exosome Isolation reagent is added at the next step, and it precipitates primarily exosomes (30-150 nm).
We have not optimized and validated a protocol to use the CD63 isolation/detection reagent with plasma samples. However, there are references in the literature that use this product in plasma samples:
Two-step magnetic bead-based (2MBB) techniques for immunocapture of extracellular vesicles and quantification of microRNAs for cardiovascular diseases: A pilot study | PLOS ONE
Yes, our Total Exosome Isolation reagents enable precipitation of the entire exosome population from cell media and all body fluids. They should also work on any kind of supernatant that contains exosomes.
We would suggest looking at the exosomes at the ultrastructural level using epon embedding, sectioning, and electron microscopy.
Yes, the exosomes recovered with the Total Exosome Isolation reagent (Cat. No. 4478359) are fully intact, free, not aggregated, and functional. They can be used for any downstream biological experiments, such as tracing them in cells or any functional studies.
The pellet is soluble in PBS or any other buffer. It is very easy to resuspend exosomes recovered from a small sample of cell culture medium (e.g., 1 mL), but if you are processing a large sample of plasma or serum, the pellet will be pretty large. In this case, you can add buffer, let it sit for 30 min at 37 degrees C, then vortex gently or pipet up and down to resuspend the exosomes.
By Nanosight analysis, most of the recovered exosomes are in the expected size range, 30-150 nm.
You can try to separate them by size, since apoptotic bodies are approximately 800-5000 nm, while exosomes are approximately 30-150 nm.
We have standardized dissolving the pellet of cell culture exosomes after isolation with Total Exosome Isolation reagent as follows:
Beckman J2-21M/E centrifuge with JA20 rotor
Nalgene centrifuge tubes for Beckman centrifuge
PBS, 0.22 µm filtered
- Start with 20-25 mL of cell culture supernatant from overnight isolation (using Total Exosome Isolation reagent).
- Centrifuge at 10,000 x g = 9200 rpm for 1 hour (set temperature at 10 degrees C, as lower setting may result in temperature as low as 0-2 degrees C during centrifugation).
- Remove supernatant by suction.
- Leave tubes upside-down on absorbant paper for 10 min at RT to remove residual buffer.
- Add buffer to cover the pellet when the tubes are placed at an angle to the bench (500 µl PBS/sample tube), leave for 30 min at RT.
- Carefully resuspend by pipetting and pool sample from each centrifuge tube.
- Collect residual volumes from each sample tube by centrifugation for 8 min at 350 x g.
- Aliquot and freeze at -80 degrees C.
Exosomes can be processed for electron microscopy for ultrastructural analysis both prior to Dynabeads magentic beads isolation and after isolation.
Prior to isolation the exosomes pool can be immunolabeled and processed for negative stain prior to ultrastructural analysis. Such a protocol could look like this:
- Load exosomes undiluted at RT for 15 min.
- Block with 0.5% BSA for 10 min.
- Label with primary antibody for 30 min.
- Wash 5X with PBS for 10 min total.
- R&M (1:100) for 30 min.
- Wash 5X with PBS for 10 min total.
- Prot A Au 10nm for 15 min.
- Wash 5X with PBS for 10 min total.
- Wash 5X with water for 10 min total.
- Embed in 0.3% uranyl acetate in methyl cellulose.
- Conduct TEM analysis.
Subpopulations of exosomes prepared using the Total Exosome Isolation kit or ultracentrifugation can also be processed for ultrathin sectioning and electron microscopy. After isolation and washing of the exosomes on the surface of the Dynabeads magnetic beads, they can be processed using the traditional TEM protocol described by Pedersen et. al. in J Virol (1999) 73:2016–2026. In brief, for conventional Epon embedding and sectioning, Dynabeads magnetic beads with exosomes can be fixed for 1 hr in 1% glutaraldehyde in 200 mM cacodylate buffer (pH 7.4), washed repeatedly in aqua destillata, and incubated for 1 hr in cacodylate buffer containing 1% OsO4 and 1.5% K3Fe(CN)6. Following two subsequent 30-min incubations in 1% tannic acid and 1.5% magnesium uranyl acetate, the samples are dehydrated by using ethanol and embedded in Epon. Ultrathin sections can be stained with lead citrate.
While we have not tested this, the Total Exosome Isolation reagent for use with cell culture medium should work, since tears are similar to cell culture medium in many ways (e.g., non viscous, with low exosome content).
Since exosomes are in the same size range as many viruses, visualization of exosomes by TIRF microscopy should work. For ultrastructural analysis, one would need to increase the resolution and use electron microscopy.
The current definition of exosomes is sophisticated and there is no consensus in the field. Exosomes are typically defined as vesicles floating in sucrose solutions at a density of approximately 1.13 to 1.19 g/ml during ultracentrifugation-based isolation. Expected size is 30-120 nm, based on the electron microscopy analysis. Protein markers include: tetraspanins (CD63, CD81, CD9), and some others. But everyone agrees that at the moment we don‘t have enough knowledge and appropriate tools to set a clear and simple definition of exosomes and other micro/nanovesicles.
There are some variations to the ultracentrifugation protocols, not based on the cell lines used so much as the experience in that particular lab, including what G-force they recommend, duration of ultra-centrifugation, straight sedimentation vs cushion vs sucrose gradients to obtain the top quality exosomes at reasonable yields. We often recommend protocols developed by Clothilde: C. Thery, S. Amigorena, G. Raposo and A. Clayton “Isolation and characterization of exosomes from cell culture supernatants and biological fluids.” Curr. Protoc. Cell. Biol., Chapter 3, Unit 3: 22, 2006.
The Total Exosome Isolation reagent allows recovery of the entire exosome population from the sample, in contrast to ultracentrifugation protocols, which recover significantly less material. Thus, the pellet is larger. The purity of exosomes is very similar for cell media samples (reagent vs ultra); for more challenging samples such as serum (which are more viscous and have much higher protein content, etc.) the reagent recovers exosomes at a lower purity (few microvesicles and large protein complexes co-precipitate) compared to ultracentrifugation. Having said this, you can obtain enough material for all standard types of downstream analysis, such as qRT-PCR or Western blot with either method.
Please review the references below for examples of exosomal markers used for this purpose:
- Journal of Extracellular Vesicles (2013) 2:20360.
- EMBO J (2007) 26(5):1221–1233.
- BMC Cancer (2010) 10(1):294.
- Cell (2012) 151:1542-1556.
When you are isolating exosomes/extracellular vesicles from cell media, for example, or body fluid such as blood, the first step is gentle centrifugation, in order to get rid of cells and cell debris. Whatever remains in the supernatant is exosomes, microvesicles, and proteins. Then you can recover exosomes using the Total Exosome Isolation reagents, and, if required, obtain an ultra-pure population of CD63 positive exosomes with CD63-coupled magnetic beads (Cat. No. 10606D). You can also isolate CD9 positive exosomes using CD9-coupled magnetic beads (Cat. No. 10614D), CD81 positive exosomes using CD81-coupled magnetic beads (Cat. No. 10616D), or use magnetic beads with EpCam (Cat. No. 10618D) to target EpCam-positive exosomes. You are right that it's not trivial (at least at the moment) to capture the specific populations using some antibodies (or other affinity reagents) since there are no absolutely specific markers for exosomes or other vesicles. It's not a problem for many projects, unless you suspect your cells are “leaky” and release some components into the media. CD63, CD81, CD9, Alix, Annexin, and TSG are not strictly specific exosome markers.
Plasma is a more challenging type of sample compared to serum. It has rather high levels of clotting factors. The current serum reagent will work on plasma, and it will precipitate all exosomes. However, the preparation will have some contaminating proteins and microvesicles, which will work for some projects, but not be acceptable for others. We recommend using our specifically optimized kits for recovery of exosomes from blood plasma (Cat. No. 4484450), as well as urine (Cat. No. 4484452), or other body fluids (Cat. No. 4484453).
The reagent for exosome isolation from serum can be used with serum from any species in addition to human. When working with small volumes of non-human samples (e.g., mouse) or when it is desired to maximize the recovery of exosomes, we recommend following the standard protocol except for spinning the samples (after incubation with the reagent) to precipitate the exosomes at 4 degrees C.
Storage should be at 2 to 8 degrees C. Freezing or drying of the Dynabeads magnetic beads is not recommended. Provided the Dynabeads magnetic beads are stored correctly, quality is guaranteed until the expiry date stated on the label.
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The binding capacity of streptavidin-coupled Dynabeads magnetic beads is fragment length-dependent. Reduced binding capacity for large DNA fragments may be due to steric hindrance. For large DNA fragments (greater than 2 kb in size), we recommend using Dynabeads kilobaseBINDER Kit.
-Salt concentration affects the binding efficiency of biotinylated nucleic acids to the Streptavidin-coupled Dynabeads magnetic beads. Optimal binding conditions for biotinylated DNA fragments (up to 1 kb) are achieved at 1 M NaCl (final concentration), 25 degrees C and 15 min incubation time. Longer DNA fragments should be immobilized overnight. Biotinylated antibodies should be immobilized in PBS buffer pH 7.4, supplemented with 0.1% BSA.
-Ensure that your sample does not contain an excess of free biotin, as the free biotin will bind Streptavidin-coupled Dynabeads magnetic beads much more rapidly than larger biotinylated molecules. Biotinylated oligonucleotides should be recovered by reverse phase HPLC or FPLC to remove free biotin from the sample.
-We also recommend a titration to optimize the quantity of beads used for each individual application, since both the size of the specific molecule to be immobilized and the biotinylation procedures affect the binding capacity of the beads.
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Yes. See this poster (https://tools.thermofisher.com/content/sfs/posters/Exosome-poster-ISEV-2013-Boston.pdf).
In addition, here are some citations:
- Blood 91:2573 (1998)
- Science 289:444 (2000)
- J Physiol 537:537 (2001)
- Mol Cell Proteomics 12:587 (2013)
- Biol Reprod 81:717 (2009)
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Yes, exosomes isolated with different surface markers can be distinctive in their protein profile. This has been demonstrated by Tauro et al. (http://www.ncbi.nlm.nih.gov/pubmed/23230278), who isolated two distinctive populations of exosomes based on surface markers EpCam or A33 from conditioned cell culture medium from a human carcinoma cell line. This proteomics study indicated that these two populations of exosomes are unique.
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We have exosome isolation kits for Exosome-Human CD63 (Cat. No. 10606D), Exosome-Human CD9 (Cat. No. 10614D), Exosome-Human CD81 (Cat. No. 10616D), and Exosome-Human EpCAM intended for isolating exosomes with these commonly used exosome surface antigens. If you are interested to isolating exosomes with other specific surface markers using your own antibody, you can use our Dynabeads exosome immunoprecipitation (Protein A, Cat. No. 10610D), Dynabeads exosome immunoprecipitation (Protein G, Cat. No. 10612D), or Exosome-Streptavidin for isolation/detection (Cat. No. 10608D). In addition, anti-mouse IgG Dynabeads magnetic beads (Cat Nos. 11031 or 11033) also can be employed in exosome isolation using mouse monoclonal antibodies against selected surface markers.
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Exosomes are usually characterized by flow cytometry (using surface markers such as CD9, CD63, TSG101, and Alix), by EM to study morphology and size, or by detailed protein analysis by LC-MS/MS.
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It depends on the cell source from which the exosomes were derived. The most commonly used surface markers for isolating and characterizing exosomes are CD9, CD63, CD81, or TSG101. Here are some of the recent references and surface markers for identifying or isolating exosomes:
Alix, CD63, EpiCam, HSP70, TSG101 Mol Cell Proteomics 12:587 (2013)
CD9, CD63 Hum Mol Genet 21:R125 (2012)
CD63, MHC IIJ Biol Chem 278:52347 (2003)
CD9, CD81, Lamp1, TSG101 Cancer Res 67:7458 (2007)
CD63 Nature Cell Biol 9:654 (2007)
Alix, CD37, CD53, CD63, CD81, CD82, TSG101J Cell Biol 200:373 (2013)
CD59, CD63, CD133, TSG101 FASEB J 23:1858 (2009)
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Exosomes can be isolated by ultracentrifugation or density gradient separation in addition to a precipitation approach. Exosomes can also be isolated by a magnetic approach using Dynabeads magnetic beads targeting exosome markers such as Human CD9, CD63, CD81, EpCAM or secondary antibody-coated Dynabeads magnetic beads using different antibodies against other exosomal surface markers.
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A range of different functions have been reported such as antigen presentation, apoptosis, angiogenesis, inflammation, and coagulation by protein/lipid exchange or activation of a signaling pathway. Exosomes provide a novel vehicle for genetic exchange between cells and mediate cell to cell communication. Exosomes also transport and propagate of infectious cargoes such as prion and retrovirus.
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Exosomes are small, membrane-bound ovoid to cup shaped particles around 30-150 nm in size containing mRNA, microRNA, proteins, and lipids. Exosomes are released by normal, abnormal, or neoplastic cells into body fluid such as blood, urine, saliva, and breast milk. Exosomes originate from the endocytic compartment and are released from cells as multivesicular bodies (MVB) fused with plasma membrane (J Cell Biol 200:373 (2013)).
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